The long-term guiding objective of the proposed research is increased understanding of the mechanism of action of the enzyme dihydrofolate reductase in terms of its three-dimensional molecular architecture. Dihydrofolate reductase is present in all self-reproducing living cells where it is essential for continuing DNA biosynthesis. This enzyme is the specific target of several clinically important chemotherapeutic agents of the antifolate family, including antineoplastic and antibiotics such as methotrexate, trimethoprim and pyrimethamine. Detailed in-depth understanding of the structure of this enzyme molecule, its conformational dynamics and how it functions in a range of species will aid medicinal chemists in designing more selectively active disease-specific chemotherapeutic compounds. X-ray crystallographic structure determinations will be carried out on a variety of substrate, cofactor and inhibitor complexes of human, chicken and E. coli dihydrofolate reductases. Specific aims include: 1) improving the resolution and refinement of the structure E. coli DHFR- NADP+ folate ternary complex in space group P3 2 21; 2) collecting x-ray data on and solving the structures of four other complexes of E. coli DHFR that crystallize isomorphously with the above; 3) solving the structures of three other non-isomorphous crystalline complexes of E. coli DHFR; 4) completing refinement and analysis of nine isomorphous crystal structures of chicken DHFR in space group C2.